Cellular hydration compositions

ABSTRACT

A composition interacts with a biological cell system that includes bioactive molecules with biomolecular surfaces, cellular components and water molecules with a specific density. The composition includes a biologically active component that is constructed to increase an activity of a biological cell system by increasing the hydration of one or more components of that cell system. The biologically active component may include a primary carbohydrate clathrate subcomponent that increases the H-bonded structure of water, and a secondary solute subcomponent. The biologically active component may include an inclusion complex that is made up of a clathrate component and a complex-forming compound. The clathrate subcomponent may include amyloses or cyclodextrins. There is also a beverage and a method that improves cellular hydration in an animal, such as a human.

FIELD OF THE INVENTION

The present invention generally relates to regulation of biological cellactivity, particularly cell activity dependent on hydration state. Moreparticularly, the present invention relates to a biologically activecomponent that is constructed to increase an activity of a biologicalcell system by increasing the hydration of one or more components ofthat cell system. That biologically active component may include aprimary carbohydrate clathrate subcomponent that increases the H-bondedstructure of water. The present invention relates further to delivery ofbiological compounds in vivo for modifying mammalian physiologicalactivity.

BACKGROUND OF THE INVENTION

Water molecules interact principally through hydrogen (H)-bonding andthrough alignment of dipole moments. For example, bonds betweenneighboring water molecules are reinforced, or stabilized, by alignmentof bond axes with next-adjacent water molecules. In liquid state water,such alignments propagate into the surrounding aqueous medium andestablish sub-micrometer scale molecular structure.

Examples of products and methods of using cyclodextrins as clathrates toform inclusions with bioactive guest molecules to improve solubilityand/or bioavailability of pharmaceutical compounds are described in:U.S. Pat. Nos. 7,115,586 and 7,202,233, and U.S. Patent ApplicationPublication Nos. 2004/0137625, and 2009/0227690, the completedisclosures of which are hereby incorporated by reference for allpurposes.

Examples of products and methods of using products containing clathratesthat bind hydrophobic biomolecules are described in U.S. Pat. Nos.6,890,549, 7,105,195, 7,166,575, 7,423,027, and 7,547,459; U.S. PatentApplication Publication Nos. 2004/0161526, 2007/0116837, 2008/0299166,and 2009/0023682; Japanese Patent Application JP 60-094912; Suzuki andSato, “Nutritional significance of cyclodextrins: indigestibility andhypolipemic effect of α-cyclodextrin” J. Nutr. Sci. Vitaminol. (Tokyo1985; 31:209-223); and Szejtl et al., Staerke/Starch, 27(11), 1975, pp.368-376, the complete disclosures of which are hereby incorporated byreference for all purposes.

U.S. Patent Application Publication No. 2009/0110746 describes chemicalagents which have the property of increasing aqueous diffusivity ofdissolved molecular oxygen (O₂) in the human body, wherein cyclodextrinsmay be included as secondary “carrier” components to improve thesolubility of primary pro-oxygenating agents, and wherein cyclodextrinsare not contemplated as agents to directly alter aqueous diffusivity,tissue oxygenation, water structure, or cellular hydration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chemical bond model of β-cyclodextrin, a cyclicoligosaccharide having seven α[1-4] linked glucose units.

FIG. 2 shows a structural model of cyclodextrins having an overalltoroid topology.

FIG. 3 shows a cyclodextrin structural model including the dispositionof glucosyl hydroxyl groups along the toroid rims.

FIG. 4 depicts a calculated molecular dynamic distribution of watermolecules surrounding a β-cyclodextrin molecule at 1 picosecond afterinitial contact.

FIG. 5 depicts the calculated molecular dynamic distribution of watermolecules of FIG. 4 at 1000 picoseconds after initial contact, includinga more organized open water structure.

FIG. 6 shows thresholded images of the water molecule distributionsshown in FIGS. 4 and 5.

FIGS. 7-10 show a comparison of NIR spectra derivatives, includingparticular wavelength regions, for water samples with and withoutdissolved cyclodextrins.

FIG. 11 shows a comparison of seed germination kinetics in watervariably including a cyclodextrin, an amino acid, and acyclodextrin/amino acid inclusion complex.

FIG. 12 shows a comparison of seed germination kinetics in watervariably including a cyclodextrin, a vitamin, and a cyclodextrin/vitamininclusion complex.

FIG. 13 shows a comparison of seed germination rate in water variablyincluding active components of hydration according to the presentdisclosure.

FIG. 14 shows a comparison of nematode longevity in media variablyincluding cyclodextrins as an active component of hydration according tothe present disclosure.

FIG. 15 shows a comparison of nematode longevity in media variablyincluding derivatized cyclodextrins as an active component of hydrationaccording to the present disclosure.

FIG. 16 shows a comparison of nematode longevity in media variablyincluding cyclodextrin inclusion complexes as an active component ofhydration according to the present disclosure.

FIG. 17 shows nematode mortality frequency in media with and without acyclodextrin inclusion complex included as an active component ofhydration according to the present disclosure.

FIG. 18 shows population survival curves for nematodes living in mediawith and without a cyclodextrin inclusion complex as an active componentof hydration according to the present disclosure.

DETAILED DESCRIPTION

Water structure is purposefully increased, or organized, by addition ofone or more solutes or suitable molecular aggregates whose surfaces arecapable of strongly competing with water molecules for H-bonding and/ordipole orientation. In particular, factors and agents that strengthenwater molecule interactions and increase water structure thereby alterthe hydration, or solvation, of a further molecular surface. Thus, aprimary solution additive that increases water structure may increasehydration interactions (e.g., bonding strength and kinetics) with amolecular surface of a secondary solution component, or alternativelydecrease such interactions, depending on the H-bonding surfacecharacteristics of the secondary component.

In addition, factors that modify water structure typically change theaverage distance between water molecules, and may thereby increase ordecrease water density. For example, as water temperature decreasesbelow its freezing point, H-bonding between the water moleculesovercomes the kinetic energy of the water molecules, resulting in anincrease in water structure that decreases the density of frozen waterby approximately 9%. Similarly in liquid state water, an increase in thestrength of water H-bonding increases the average distance between watermolecules, which is observed as an increase in specific volume (i.e.,decrease in density). A decrease in density of liquid water may increasethe diffusivity of a dissolved solute. Thus, an aqueous additivecomponent which decreases water density may increase the diffusivity ofa co-dissolved solute.

Chaotropes, as used herein, are aqueous solute additives that disrupthydrogen bonded networks in aqueous solutions, and thereby act todecrease water structure. Chaotropes typically are less polar and haveweaker H-bonding potentials than water molecules. Chaotropes maypreferentially bind to non-polar solutes and particles, and therebyincrease solubility of a non-polar solute.

Kosmotropes, as used herein, are solutes that promote strong andextended H-bonded networks in aqueous solutions, and which therebyincrease and/or stabilize the sub-micrometer scale structure of watermolecule interactions. A kosmotrope having an H-bonding chemicalpotential greater than that of water, and/or having a dipole momentgreater than that of water, may increase H-bonded networks between watermolecules. Further, by strengthening hydration structure, a kosmotropemay increase hydration interactions at a molecular surface, which mayinclude a binding site between molecules. A kosmotrope may thus be usedas an aqueous solution additive to stabilize molecular interactions.

Further, a kosmotrope may increase the effective chemical activity of adissolved co-solute. An increase in the strength of H-bondinginteractions between water molecules causes water to adopt a more openarchitecture having a lower specific density and higher specific volume.Thus, by causing a decrease in density, addition of a kosmotrope to anaqueous solution may increase a diffusivity of one or more of adissolved co-solute species or compound. Increasing the diffusivity of asolute species or compound may increase its reactivity, chemicalpotential, effective concentration, and availability.

As discussed herein, clathrate components are amphipathic carbohydratecompounds which have external surfaces that are hydrophilic and H-bondstrongly with water, and also internal surfaces that are lesshydrophilic. A clathrate's internal surface may selectively bind amolecular structure which is relatively non-polar or less hydrophilicthan water.

An inclusion complex, as used herein, is a chemical complex formedbetween two or more compounds, where a first compound (also referred toas a host) has a structure that defines a partially enclosed space intowhich a molecule of a second compound (also referred to as a guest) fitsand binds to the first compound. The host molecule may be referred to asa clathrate, and may bind the guest molecule reversibly or irreversibly.

A biological cell, as used herein, is the self-replicating functionalmetabolic unit of a living organism, which may live as a unicellularorganism or as a sub-unit in a multicellular organism, and whichcomprises a lipid membrane structure containing a functional network ofinteracting biomolecules, such as proteins, nucleic acids, andsaccharides. Biological cells include prokaryote cells, eukaryoticcells, and cells dissociated from a multicellular organism, which mayinclude cultured cells previously derived from a multicellular organism.

A biological cell system, as used herein, is a functionallyinterconnected network of biological cells and/or sub-cellular elements,which may include living cells, non-living cells, cellular organelles,and/or biomolecules.

A bioactive molecule, as used herein, is a molecular compound having afunctional activity in a biological cell system.

A biomolecule, as used herein, is a molecular compound that issynthesized by a biological cell. Biomolecules include compoundsnormally synthesized by cells, and compounds synthesized by geneticallyengineered cells, and chemically synthesized copies of cell-derivedcompounds.

A biomolecular surface, as used herein, is an outer atomic boundary of abiomolecule, which may include a biochemical interaction surface, suchas a binding site.

Cellular components, as used herein, are functional elements of abiological cell, which include biomolecules, biomolecule complexes,organelles, polymeric structures, membranes and membrane-boundstructures, and may further include functional pathways and/or networks,such as a sequence of molecular events.

The density of a substance is the mass per unit volume of that substanceunder specified conditions of temperature and pressure.

The specific volume of a substance is the volume per unit mass of thesubstance, which may be expressed, for example, as m³/kg. The specificvolume of a substance is equivalent to the reciprocal of the density ofthat substance.

A biologically active component, as used herein, is a molecularsubstance that modifies (increases or decreases) an activity of abiological cell system.

A bioactive agent, as used herein, is a substance that when added to abiological cell system, or to a cellular component, causes a change inthe biological activity of that system, or that component.

The bonded structure of water, as used herein, refers to the network ofH-bonds that hold and organize the orientation of water molecules inliquid and solid states. Water structure, as used herein, increases whenH-bonds between water molecules at a given temperature are strengthened,and decreases when H-bonds between water molecules at a giventemperature are weakened.

An interaction between cellular components, as used herein, refers to achemical binding between biomolecular surfaces. Such interaction mayinclude binding between two biomolecules, such as a ligand and itsspecific receptor. Alternatively, such interaction may include bindingbetween a biomolecule and an organelle, such as a cell membrane.

Extracellular signals, as used herein, are biomolecules that can modify(increase or decrease) an activity of a cell when applied to the outsideof the cell. An extracellular signal may bind to a component of thecell's plasma (outer) membrane, or alternatively may pass through theplasma membrane to regulate an intracellular activity. Extracellularsignals may include, but are not limited to, extracellular matrixcomponents; cell membrane components such as glycoproteins andglyocolipids; antigens; and diffusible biomolecules such as nitricoxide.

An intracellular messenger, as used herein, is an internal component ofa biological cell that has an active state, and which serves in anactive state as an intermediate signal to transmit an extracellularsignal to an intracellular target.

A pharmacological agent, as used herein, is a synthetic chemicalsubstance that binds to and thereby alters the activity of a biomoleculeor a biomolecule complex.

The present invention includes active compositions that increase anactivity of a biological cell system by increasing the hydration of oneor more components of that cell system.

Preferably, an active composition for modifying cellular hydrationincludes a primary carbohydrate clathrate component that increases theH-bonded structure of water. In some examples, the active compositionpreferably includes a primary carbohydrate clathrate component thatincreases the H-bonded structure of water and a secondary solutecompound, which may be a bioactive agent. In some examples, the activecomposition preferably includes an inclusion complex formed between aclathrate component and a complex-forming compound, which may be abioactive agent.

Biological cells are multi-compartment structures, comprising chemicallyactive water-based chambers and lipid-based membranes. The structure andactivity of cells derives from highly selective chemical bondingassociations between their biomolecular components, such as lipids,structural proteins, enzymatic proteins, carbohydrates, salts,nucleotides, and other metabolic and signaling biomolecules. Thestrength and specificity of biomolecule bonding reflects complementarychemical topologies at the bonding interface. Hydrophilic and/orhydrophobic surfaces commonly dominate the chemical topology ofbiomolecular bond interfaces. In aqueous systems, hydrophobic andhydrophilic interactions are substantially driven by competing hydrationinteractions with molecules of water, whose concentration exceeds 50 M.

Cellular hydration, as used herein, refers to interaction between watermolecules and biomolecular components of a cellular system. Cellularhydration may be modified by changing the strength and/or kinetics ofH-bonding between water molecules and biomolecular surfaces.

An aqueous solution additive that modifies water structure may, bymodifying the hydration of biomolecular binding surfaces, alter thestrength, kinetics, and/or specificity of binding between cellularcomponents. For example, a kosmotrope aqueous additive that increaseswater structure may alter the strength, kinetics, and/or specificity ofbinding between a secreted intercellular signaling factor and a cognatereceptor located in the plasma membrane of a potential target cell forthat factor, and hence bias the outcome of a cellular signaling network.

Clathrates that are suitable as active components of cellular hydrationaccording to the present invention include amyloses and cyclodextrins.Amyloses are linear polysaccharides of D-glucose units. As shown in FIG.1, cyclodextrins are macrocyclic oligosaccharides of D-glucose unitslinked by α(1-4) interglucose bonds. Amylose and cyclodextrin arereadily prepared in large quantities from hydrolyzed starch.Cyclodextrin preparation includes enzymatic conversion, most commonlyusing the enzyme cyclodextrin-glycosyl transferase produced by Bacillusstrains.

As shown in FIG. 2, cyclodextrins may differ by the number of glucoseunits included in the ring. Cyclodextrin species include α-cyclodextrin(6 units), β-cyclodextrin (7 units), γ-cyclodextrin (8 units), andδ-cyclodextrin (9 units). Parent cyclodextrins, as used herein, arenatural, chemically underivatized α- β- and γ-cyclodextrins, having 18(α-), 21 (β-) and 24 (γ-) free, unmodified hydroxyl groups,respectively.

As schematically shown in FIG. 3, cyclodextrins have a toroid topology,a shape which generally resembles a truncated cone, or half of anopen-ended barrel. Accordingly, a cyclodextrin may be described asincluding an exterior chemical surface, which includes the outer surfaceand the rims of the barrel, and an interior chemical surface surroundingan internal cavity (the inside of the barrel).

Cyclodextrin exterior surfaces include a high density of hydrophilicchemical groups that H-bond with water. In particular, the hydroxylgroups of the parent α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrinstructures are all concentrated at the ends of the cyclodextrin barrel.More particularly, cyclodextrin hydroxyl (—OH) chemical groups arelocated along the barrel rims, and their orientation is stericallyrestricted. Hydroxyl groups at glucose position C(6), which may becalled primary OH groups, point in a counter-clockwise direction withrespect to the narrower open end of the cyclodextrin barrel. Hydroxylgroups at glucose position C(2), which may be called secondary hydroxylgroups, angle in a clockwise direction with respect to the wider openend of the cyclodextrin barrel.

The high density and constrained orientation of cyclodextrin hydroxylgroups creates particularly strong H-bonding surfaces at both ends ofthe cyclodextrin barrel. Physicochemical analysis and solvation modelingof cyclodextrins show water molecules adjacent the cyclodextrin havefixed positions and low angular (rotational) mobility. Usefully, speciesof cyclodextrin, which differ in barrel diameter as well as number ofhydroxyl groups, also differ in the number and mobility of stronglybound water molecules.

The H-bonding activity of a cyclodextrin compound may propagate into asurrounding aqueous medium. As shown in FIGS. 4 and 5, dynamic modelingof a cyclodextrin molecule introduced into a defined population of watermolecules at standard temperature and pressure causes a nanosecondreorganization of water throughout the volume. FIG. 4 depicts apopulation distribution at one picosecond (ps) after initiating themixing simulation; FIG. 5 depicts a redistribution of the samepopulation at 1000 ps (1 nanosecond), wherein water molecules haveadopted a more open structure.

In some examples, a cyclodextrin may function as an active component ofcellular hydration through a kosmotrope activity that increases thebonded structure of water, wherein an increase in H-bonding betweenwater molecules modifies the hydration of biomolecular surfaces, andthereby alters the strength, kinetics, and/or specificity of bindingbetween cellular components.

In some examples, a cyclodextrin may function as an active component ofcellular hydration through a kosmotrope activity that increases thebonded structure of water, wherein stronger H-bonding between watermolecules causes an open water structure having a lower specific density(i.e., a higher specific volume), and wherein a rate of diffusion ofbioactive molecules is increased. Such examples may include a solublebioactive molecule such as an enzyme, enzyme substrate, nutrient,metabolite, cytokine, neurotransmitter, hormone, extracellular signal,intracellular messenger, or pharmacological agent.

An active component of cellular hydration that increases a rate ofdiffusion in water may regulate one of the many biological processesthat are limited by the rate of change in the concentration of abioactive component. For example, clearance of a neurotransmitter fromsynaptic clefts is commonly diffusion limited, including the passivedispersal of glutamate from excitatory synapses in the mammalian brain,and the active catabolism of acetylcholine at vertebrate neuromuscularsynapses by the diffusion-limited enzyme acetylcholine esterase.Similarly, the activity of electrically excitable cells, such as musclecells, is commonly coordinated by the diffusion-limited changes in theconcentration of the intracellular second messenger signal calcium.

The cellular hydration activity of a cyclodextrin may be modified,either increased or decreased, by forming an inclusion complex with acomplex-forming agent. Internal surfaces of cyclodextrins lack hydroxylgroups, are less hydrophilic than the surrounding aqueous environment,and thereby preferentially bind co-solute molecules having lowhydrophilic and H-bonding potential.

Upon ingestion by an animal, carbohydrate clathrate compositions thatincrease the hydrogen bonding structure of interstitial andintracellular fluids may improve cellular hydration, including hydrationstructure at cell membrane surfaces as well as solvation of biomoleculesthat sub-serve healthy cell function. Improved cellular hydration maysupport healthy cell function by, for example, increasing the import,export, and/or diffusivity of solutes, nutrients, waste products,cytokines, metabolites, and other molecular agents supportive of cellfunction, differentiation, repair, growth, and survival, and bystabilizing cellular membranes in vulnerable tissues, such as muscle andnerve.

In some examples, a carbohydrate inclusion complex ingested by an animalmay increase water H-bonding structure and thereby improve cellularhydration and/or diffusivity of cellular components. In some examples, acarbohydrate inclusion complex ingested by an animal may dissociate torelease a free (i.e., non-complexed) cyclodextrin clathrate componentthat increases water hydrogen-bonding structure and thereby improvescellular hydration and/or diffusivity of cellular components. In someexamples, a carbohydrate inclusion complex may increase water structureand improve cellular hydration without dissociating. In some examples, acarbohydrate inclusion complex may dissociate into a clathrate componentfor increasing water structure and cellular hydration, and acomplex-forming agent which may further increase water-structure and/orprovide other beneficial properties, such as nutrition or flavor.

The carbohydrate clathrate compositions of the present invention may beprovided in various forms, including being formed into a solid powder,tablet, capsule, caplet, granule, pellet, wafer, powder, instant drinkpowder, effervescent powder, or effervescent tablet. Some carbohydrateclathrate compositions may also be formed as, or incorporated into,aqueous beverages or other food products. Such carbohydrate clathratecompositions may be inclusion complexes that remain reasonably stableduring storage, so that the clathrate component does not dissociate fromthe complex-forming agent and form a stronger complex with anothercompound that reduces the kosmotropic activity of the complex andthereby decrease its ability to improve cellular hydration.

The present disclosure also provides methods for improving cellularhydration in an animal, such as a human. For example, some methods mayinclude (a) preparing a beverage by dissolving an inclusion complexformed by a carbohydrate clathrate component and a complex-forming agentcapable of dissociating from carbohydrate clathrate component underphysiological conditions, and (b) having the animal orally ingest thebeverage, whereupon the carbohydrate clathrate component dissociatesfrom the complex-forming agent and modifies the strength, extent, andkinetics of the hydrogen bonded water structure at cellular biomolecularsurfaces.

I. CARBOHYDRATE CLATHRATE COMPOSITION

The carbohydrate clathrate component may include any suitablecarbohydrate including, but not limited to, α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, methylated β-cyclodextrins,2-hydroxypropylated β-cyclodextrins, water soluble β-cyclodextrinpolymers, partially acetylated α-, β-, and γ-cyclodextrins, ethylatedα-, β-, and β-cyclodextrins, carboxy-alkylated β-cyclodextrins,quaternary-ammonium salts of α-, β-, and γ-cyclodextrins, an amylose(e.g., an acetylated amylose), and mixtures thereof.

In preferred embodiments, the carbohydrate clathrate may be selectedbased upon a kosmotrope activity that increases water structure alone orin combination with other solutes. Preferred cyclodextrin kosmotropesmay include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,2-hydroxypropyl-cyclodextrins, carboxymethylated-cyclodextrins, andquaternary-ammonium-cyclodextrins.

Cyclodextrin derivatives may include alkylated, hydroxyalkylated,alkoxyalkylated, acetylated, quaternary ammonium salts,carboxyalkylated, maltosylated, and glucosylated derivatives. Alkylgroups of cyclodextrin derivatives may be straight chain or branched,may have main chain lengths of one to three carbons, and may have atotal of one to six, and preferably one to three carbon atoms. Somenon-limiting examples of cyclodextrin derivatives may include methylatedbeta-cyclodextrins, 2-hydroxypropylated β-cyclodextrins, water solublebeta-cyclodextrin polymers, partially acetylated α-, β-, and/orγ-cyclodextrins, ethylated α-, β-, and/or γ-cyclodextrins,carboxyalkylated β-cyclodextrins, quaternary ammonium salts of α-, β-,and/or γ-cyclodextrins, as well as mixtures of any combination of thesederivatives, together or in combination with one or more cyclodextrins.An exemplary mixture of cyclodextrins may include a combination of α-,β-, and/or γ-cyclodextrin in a weight ratio range of about 1:1:1 to2:2:1, respectively. The cyclodextrin may be in a hydrate crystallineand/or amorphous form, including but not limited to the hydrate and/oramorphous forms of α-, β-, and/or γ-cyclodextrin, and mixtures thereof.

If the carbohydrate clathrate composition is in solid form, thecyclodextrin component may be present in a concentration range of about10-90% w/w, or about 15-70% w/w, or about 15-60% w/w. Preferably, thecyclodextrin component may be present in a concentration range of about10-50% w/w, or about 15-40% w/w. More preferably, the cyclodextrincomponent may be present in a concentration range of about 20-25% w/w.

If the carbohydrate clathrate composition is in the form of an aqueousbeverage, the cyclodextrin component may be present in a concentrationrange of about 0.01-75% w/v, or about 0.05-50% w/v, or about 0.1-25%w/v. Preferably, the cyclodextrin component may be present in aconcentration range of about 0.1-10% w/v. More preferably, thecyclodextrin component may be present in a concentration range of 0.1-5%w/v.

The carbohydrate clathrate composition may preferably include aclathrate capable of forming an inclusion complex with a variety ofcomplex-forming agents, such as amino acids, vitamins, flavorants,odorants, colorants, and the like. Non-exclusive examples ofcarbohydrate clathrate components capable of binding a complex-formingagent to form an inclusion may include α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, 2-hydroxypropyl-cyclodextrins,caboxymethylated-cyclodextrins, quaternary-ammonium-cyclodextrins,amyloses, amylose derivatives, or any desired mixture of these.

A cyclodextrin clathrate component may be further selected based uponits desired binding properties with selected complex-forming agents.Non-limiting examples of acceptable cyclodextrins may includecommercially available and government regulatory approved forms of α-,β- and γ-cyclodextrins. The number of glucose units determines theinternal dimensions of the cavity and its volume, and may determine aselectivity in forming inclusion complexes with a guest molecule.Selected complex-forming agents, when bound to a host cyclodextrin orother host carbohydrate clathrate, may modify the physico-chemicalproperties of the complexed host to increase its kosmotropic activity.

If the clathrate component is in the form of an amylose component, theamylose component may contain glucose units expressed as degree ofpolymerization (DP) in the range of DP=10-900, and more preferablyDP=20-200, and most preferably DP=30-80. Amylose derivatives mayinclude, but are not limited to, acetylated amyloses. The amylosecomponent preferably may have a structure that includes α1,4-linkedD-glucopyranoses in a helical arrangement that defines a central cavityfor binding hydrophobic molecules. For example, the A- and B-starchhelix of V-amylose may include a parallel, left-handed double helixdefining a central cavity. The helices of amylose inclusion complexesmay be stabilized by the hydrophobic forces created by the host-guestinteractions, intermolecular H-bonds between glucoses in adjacentamyloses, and intramolecular H-bonds formed by adjacent turns of thehelix. See Hinrichs, W., et al., “An Amylose Antiparallel Double Helixat Atomic Resolution,” Science, (1987), 238(4824): 205-208, the completedisclosure of which is hereby incorporated by reference for allpurposes. An amylose clathrate component maybe used to form an inclusioncomplex with a complex-forming agent having a low molecular weight, suchas the non-limiting examples of flavorants, colorants, vitamins, aminoacids, and/or amines.

If the composition containing an amylose clathrate component is in solidform, the amylose component preferably may be present in a concentrationrange of about 10-90% w/w, or about 15-70% w/w, or about 15-60% w/w.More preferably, the amylose component may be present in a concentrationrange of about 10-50% w/w, or about 15-40% w/w. Most preferably, theamylose component may be present in a concentration range of about20-25% w/w. If the composition containing the amylose clathratecomponent is in the form of an aqueous beverage, the amylose componentpreferably may be present in a concentration range of about 0.1-75% w/v,or about 1-50% w/v, or about 1-25% w/v.

II. COMPLEX FORMING AGENT

In some examples, the clathrate compositions disclosed herein mayoptionally contain a complex-forming agent, which may include one ormore amino acids, vitamins, flavorants, odorants, and/or othernutritional components, as well as combinations or mixtures of theseagents. The carbohydrate clathrate compositions may further include oneor more carbonation forming components for use in forming beverageproducts.

The complex-forming agents may strongly complex with the clathratecomponent so as to increase a kosmotropic activity and thereby influencecellular hydration. Alternatively, these agents may weakly complex withthe clathrate component so as to have the capability of dissociatingtherefrom in order to allow a free clathrate component to increase waterstructure.

Non-limiting examples of amino acids suitable for forming inclusioncomplexes with the carbohydrate clathrate compositions of the presentdisclosure may include aspartic acid, arginine, glycine, glutamic acid,proline, threonine, theanine, cysteine, cystine, alanine, valine,tyrosine, leucine, isoleucine, asparagine, serine, lysine, histidine,ornithine, methionine, carnitine, aminobutyric acid (alpha-, beta-, andgamma-isomers), glutamine, hydroxyproline, taurine, norvaline,sarcosine, salts thereof, and mixtures thereof. Also included areN-alkyl C₁-C₃ and N-acylated C₁-C₃ derivatives of these amino acids, andmixtures of any of the amino acids or derivatives thereof. Preferredcomplex forming amino-acids that may be included with cyclodextrins toincrease water structure and cellular hydration include L-arginine,L-lysine, N-methyl-lysine, and L-carnitine.

Non-limiting examples of vitamins may include nicotinamide (vitamin B₃),niacinamide, niacin, pyridoxal hydrochloride (vitamin B₆), ascorbicacid, edible ascorbyl esters, riboflavin, pyridoxine, thiamine, vitaminB₉, folic acid, folate, pteroyl-L-glutamic acid, pteroyl-L-glutamate,salts thereof, and mixtures thereof. Preferred vitamins included withcyclodextrins to increase water structure and cellular hydration mayinclude nicotinamide and niacinamide.

Non-limiting examples of flavorants may include apple, apricot, banana,grape, blackcurrant, raspberry, peach, pear, pineapple, plum, orange,and vanilla flavorants. Examples of flavorant related compounds includebutyl acetate, butyl isovalerate, allyl butyrate, amyl valerate, ethylacetate, ethyl valerate, amyl acetate, maltol, isoamyl acetate, ethylmaltol, isomaltol, diacetyl, ethyl propionate, methyl anthranilate,methyl butyrate, pentyl butyrate, and pentyl pentanoate. A flavorant maybe selected so that it weakly binds to a selected cyclodextrin componentwith a binding constant in the range of about 10 to 800 M⁻¹, preferably30 to 150 M⁻¹, and more preferably 40 to 100 M⁻¹.

Non-limiting examples of other taste improving components may includepolyol additives such as erythritol, maltitol, mannitol, sorbitol,lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol(glycerine), threitol, galactitol, palatinose, reducedisomalto-oligosaccharides, reduced xylo-oligosaccharides, reducedgentio-oligosaccharides, reduced maltose syrup, and reduced glucosesyrup.

Non-limiting examples of colorants may include those that are known tobe more water soluble and less lipophilic. Examples of colorants withthose properties are betalains, such as betacyanins and betaxanthins,including vulgaxanthin, miraxanthin, portulaxanthin and indicaxanthin;anthocyanidins, such as aurantinidin, cyanidin, delphinidin,europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidinand rosinidin, as well as all corresponding anthocyanins (or glucosides)of these anthocyanidins; and turmeric type colorants including phenoliccurcuminoids, such as curcumin, demethoxycurcumin andbisdemethoxycurcumin.

All of the above examples of amino acids, vitamins, flavorants andrelated compounds may be in appropriate salt or hydrate forms.

The complex-forming agent may be selected to form an inclusion complexwith a selected clathrate component. The complex-forming agent may bindto the clathrate component as a guest molecule in the cavity of theclathrate molecule, and/or may form a so-called outer sphere complex,where the selected weak complex-forming agent binds to the clathratemolecule at a position at or around the rim(s) of the clathrate. Forexample, the selected weak complex-forming agent may be bound to acyclodextrin molecule at or around the primary and/or secondary hydroxylgroups at the rims of the cyclodextrin torus. Some complex-formingagents that form an outer sphere complex with the selected cyclodextrinmay reduce or prevent self-aggregation of dissolved, hydratedcyclodextrin molecules by masking intermolecular hydrogen bonds thatform between two neighboring cyclodextrin molecules in water.

If the carbohydrate clathrate composition is in solid form, thecomplex-forming agent may be present in a concentration range of about1-50% w/w. Preferably, the complex-forming agent may be present in aconcentration range of about 1-40% w/w or about 1-25% w/w. Morepreferably, the complex-forming agent may be present in a concentrationrange of about 5-15% w/w.

If the carbohydrate clathrate composition is in the form of an aqueousbeverage, the complex-forming agent may be present in a concentrationrange of about 0.1-25% w/v or about 1-20% w/v. Preferably, thecomplex-forming agent may be present in a concentration range of about1-15% w/v or about 1-10% w/v or about 3-8% w/v. More preferably, thecomplex-forming agent may be present in a concentration range of about5-8% w/v.

III. THE INCLUSION COMPLEX

As noted above, the inclusion complex may include a clathrate hostmolecule complexed with one or more complex-forming agents. In the formof a solid product, such as a solid powder or tablet, the inclusioncomplex may exhibit some unique properties as compared to a solidcomposition containing essentially the same components, but without thepreliminary formation of the inclusion complex. The inclusion complex isessentially a chemical entity having non-covalent hydrogen bonds formedbetween the clathrate molecule and the weak complex-forming agentmolecule. The inclusion complex, in its solid form, has the potential ofdissociating into the clathrate component for increasing water structureand the complex-forming agent, which may further increase waterstructure or provide other beneficial properties, such as nutrition orflavor, when the inclusion complex is introduced to an aqueousenvironment, such as upon dissolution in an aqueous beverage, or uponingestion.

When in the form of a solid product, the clathrate component and one ormore types of a complex-forming agent may be substantially in the formof an inclusion complex, as described above. Preferably, over about 25%of the clathrate component is complexed with one or more types of acomplex-forming agent in the form of an inclusion complex. It isprogressively more preferable to have over 35%, 45%, 50%, 60%, 70%, 80%,90%, and 95% of the clathrate component complexed.

IV. CARBONATION-FORMING COMPONENTS

Some clathrate compositions may include carbonation-forming componentsthat produce carbonation, or effervescence, upon dissolution into anaqueous environment. Carbonation-forming components advantageously mayinhibit self-aggregation of clathrate molecules, thereby increasingclathrate surface area for structuring water and increasing cellularhydration.

Non-limiting examples of carbonation-forming components may includesodium carbonate, sodium bicarbonate, potassium carbonate and potassiumbicarbonate. Preferred carbonation-forming components may include sodiumcarbonate, and sodium bicarbonate.

If the carbohydrate clathrate composition is in solid form, thecarbonation-forming component may be present in a concentration range ofabout 1-60% w/w or about 5-60% w/w. Preferably, the carbonation-formingcomponent may be present in a concentration range of about 5-45% w/w or10-45% w/w. More preferably, the carbonation-forming component may bepresent in a concentration range of about 10-15% w/w.

If the carbohydrate clathrate composition is in the form of an aqueousbeverage, the carbonation-forming component may be present in aconcentration range of about 1-30% w/v or about 1-25% w/v. Preferably,the carbonation-forming component may be present in a concentrationrange of about 2-15% w/v or 2-10% w/v. More preferably, thecarbonation-forming component may be present in a concentration range ofabout 2-5% w/v.

V. OTHER COMPONENTS

Some compositions may include yet other components that affect the tasteand/or nutritional value of the composition. These additional componentsmay include, but are not limited to, one or more of the following:flavor additives, nutritional ingredients and/or various hydroxyl-acidsthat act as clathrate aggregation-preventing additives in theformulations. Non-limiting examples of such other components may includecitric acid, ascorbic acid, sodium chloride, potassium chloride, sodiumsulfate, potassium citrate, europium chloride (EuCl₃), gadoliniumchloride (GdCl₃), terbium chloride (TbCl₃), magnesium sulfate, alum,magnesium chloride, maltodextrin, mono-, di-, tri-basic sodium orpotassium salts of phosphoric acid (e.g., inorganic phosphates), saltsof hydrochloric acid (e.g., inorganic chlorides), sodium bisulfate.Non-limiting examples of hydroxyl-acids that prevent cyclodextrinaggregation may include isocitric acid, citric acid, tartaric acid,malic acid, threonic acid, salts thereof and mixtures thereof. Thesehydroxyl-acids also may exhibit some nutritional benefits. Othernon-limiting examples of additional optional components, such as tasteadditives, that may be used include suitable organic salts, such ascholine chloride, alginic acid sodium salt (sodium alginate),glucoheptonic acid sodium salt, gluconic acid sodium salt (sodiumgluconate), gluconic acid potassium salt (potassium gluconate),guanidine HCl, glucosamine HCl, amiloride HCl, monosodium glutamate(MSG), adenosine monophosphate salt, magnesium gluconate, potassiumtartrate (monohydrate), and sodium tartrate (dihydrate).

Preferred other components may include, for example, citric acid,ascorbic acid, and maltodextrin.

If the carbohydrate clathrate composition is in solid form, the one ormore other components each may be present in a concentration range ofabout 1-30% w/w or about 1-25% w/w. Preferably, the one or more othercomponents each may be present in a concentration range of about 1-20%w/w or 1-15% w/w. More preferably, the one or more other components eachmay be present in a concentration range of about 2-5% w/w.

If the carbohydrate clathrate composition is in the form of an aqueousbeverage, the one or more other components may be present in aconcentration range of about 1-20% w/v or about 1-15% w/v. Preferably,the one or more other components may be present in a concentration rangeof about 1-10% w/v or 1-5% w/v. More preferably, the one or more othercomponents may be present in a concentration range of about 1-3% w/v.

VI. COMPONENT RATIOS

In addition to the above descriptions regarding the types and amounts ofthe various components that may be employed in the carbohydrateclathrate compositions disclosed herein, it is additionally noted thatthe relative amounts of these components can be described as well.Preferably, the weight ratio of the clathrate component to thecomplex-forming agent may be in the range of about 5:1 to 1:10, morepreferably may be in the range of about 2:1 to 1:5, still morepreferably may be in the range of about 2:1 to 1:2, and yet morepreferably may be in the range of about 1:1 to 1:2.

Regarding the other possible components, such as flavor components,carbonation-forming components, and other components described above,the weight ratio of the clathrate component to each of the othercomponents separately may be in the range of about 25:1 to 1:25, orabout 10:1 to 1:10, or about 5:1 to 1:5, or optionally about 2:1 to 1:2,as well as 1:1.

VII. PREFERRED EMBODIMENTS

Preferred embodiments of the carbohydrate clathrate compositiondisclosed herein are provided as illustrations, and are not intended tolimit the scope of this disclosure in any way.

Example 1 Effect of Cyclodextrin on Molecular Dynamics of WaterStructure

A simulated water solvated cyclodextrin molecular system was createdusing HyperChem® 5.11 software (from HyperCube Inc, Gainesville, Fla.),with input parameters derived from single crystal analysis ofcyclohepta-amylose dodecahydrate clathrate (or, β-cyclodextrin) reportedby Lindner and Saenger (see: Carbohydr. Res., 99:103, 1982), and using awater periodic solvent box (3.1×3.1×3.1 nm³) containing altogether 984water molecules. Molecule conversion and atom type were adjusted to theproper format using TinkerFFE 4.2 (TINKER Software Tools for MolecularDesign, Version 5.0, Jay William Ponder, Washington University, St.Louis, Mo.). Molecular mechanics and dynamics calculations wereperformed with Tinker 5.0 software after preliminary optimization of thetruncated Newton-Raphson method using a Linux x86-64 operating system(S1amd 64 v12.2).

Molecular dynamics simulations were run using MM3 Force Field molecularmechanics software, at constant temperature (298 K) for 120 picosecond(psec), with 0.1 femtosecond (fsec) steps. Recordings were generated bydumping intermediate structures every 100,000 steps (equivalent to 10psec elapsed time).

Observations:

At time zero of each simulation, the standard water solvent boxcontained one β-cyclodextrin clathrate molecule and a uniformlydistributed population of 984 water molecules. FIGS. 4 and 5 show arepresentation of the central portion of the solvent box at particularelapsed times during one representative simulation. It will beappreciated that water molecule positions and orientations arerepresented as (bent) rods, while β-cyclodextrin is represented as a vander Waals surface. It will be further appreciated that FIGS. 4 and 5depict a volume of the solvent box, and therefore compress a threedimensional molecular distribution into two dimensions.

FIG. 4 shows a central portion of the solvent box at 1 psec of elapsedtime of a simulation. In particular, at 1 psec of elapsed time, watermolecules immediately adjacent to β-cyclodextrin have acquiredrelatively static (stable) positions through H-bonding to cyclodextrin.Such water molecules may be referred to as a first hydration layer.However, the distribution of most water molecules in the solvent boxremains generally similar to the starting distribution (1 psecprevious), which is unstructured.

FIG. 5 shows the simulation of after 1000 psec (i.e., 1 nsec) of elapsedtime. At 1000 psec, water molecules immediately adjacent toβ-cyclodextrin continue to occupy relatively static (stable) positions.However, compared to 1 psec (FIG. 4), water molecules beyond the firsthydration layer have acquired a more open microstructure.

Differences in water structure may be more readily observed in theabsence of the perspective shadowing detail included in FIGS. 4 and 5.FIG. 6 shows alternative views of the water molecule distributions shownin FIG. 4 (left side, labeled 1 psec) and FIG. 5 (right side, labeled1000 psec), which were produced by the following methods: image filesfor FIGS. 4 and 5, having 256 grey levels (8 bits), were opened inPhotoshop 9.0 (Adobe, Inc), adjusted to 300 dpi, thresholded at greylevel 207; images were cropped to an identical outer annulus diameterusing the circle select tool, and the outer square corners filled withblack (grey level 0), and then further cropped to blacken an innerannulus that barely includes the cyclodextrin molecule. The dimensionsof the outer and inner annuli are identically applied to the comparedimages. The resulting thresholded representations qualitatively showwater molecules surrounding the central (occluded) cyclodextrin moleculehave a more open and coordinated structure at 1000 psec (e.g., rightside panel of FIG. 6).

To quantitatively assess the change in microstructure of waterrepresented in FIGS. 4-6, molecular density was approximated bymeasuring open paths through the depicted volume, a method similar to amean free path analysis, where a mean free path in a defined volume of amolecular substance is inversely related to the density of themolecules. In particular, an open path between water molecules is shownby a white pixel element, and the number of open paths in the volume isreadily quantitated using the histogram tool of Photoshop 9.0 to countthe number of white pixel elements. Applied to the panels of FIG. 6, ameasured increase of 2% was calculated for open paths at 1000 psec ofelapsed time compared to open paths at 1 psec of elapsed time. Forcomparison, freezing of pure water results in a 9% decrease in density.As path length is inversely proportional to molecule density, theanalysis indicates that dissolved cyclodextrins decrease the density ofan aqueous solution by increasing the organization of water molecules.

In summary, the results indicate a rapid (psec) H-bonding adhesionbetween the outer surface hydroxyls of β-cyclodextrin and watermolecules is followed by a slower (nanosecond) propagation of watermolecule reorientation throughout the solvent box, resulting in a moreopen water structure. The measured results further indicate that acyclodextrin may sufficiently increase H-bonding between water moleculesin the surrounding aqueous volume to result in a decrease in the densityof water.

Example 2 Effect of Cyclodextrin Additives on Water Bonding Detected byIR Spectroscopy

Physical micro-structure studies of water, water-sugar interactions, anddetection of sugar effects on increasing and decreasing water structurehave preferentially employed infrared (IR) spectroscopy, andparticularly near infrared (NIR) spectroscopy, as for example reportedby Segtan et al. (see: Anal. Chem. 2001; 73, 3153-3161), and R.Giangiacomo (see: Food Chemistry, 2006, 96.3. 371-379.)

Hydration bond energies in pure waters and solutions of the same waterscontaining cyclodextrin compounds were assayed using IR spectroscopy inthe near and middle infrared ranges. To record linear signals throughoutan entire wavelength range, attenuation from water absorbance wasminimized with a short optical length cuvette.

NIR range spectra were registered on a FOSS NIR Systems, Inc. 6500spectrometer and Sample Transport Module (STM) using a 1 mm-es cuvette.Transmission spectra were collected from 1100-2498 nm using a leadsulfide (PbS) detector and Vision 2.51 software (2001; FOSS NIRSystems,Inc.)

A Perkin-Elmer Spectrum 400 FT-NIR/FT-IR spectrometer and UATR(Universal Attenuated Total Reflectance; ZnSe-diamond crystal, 1× flattop plate) sample handling unit were used to obtain spectra across2500-15385 nm (reported as 4000-650 cm⁻¹). Measurements were performedat 24 C using a triglycine-sulfate (TGS) detector and Spectrum ES 6.3.2software (PerkinElmer, 2008).

Three samples of water were used in the present study. A first watersample (USA I) was obtained from the U.S. and was purified by reverseosmosis, carbon filtration, ultraviolet light exposure, membranefiltration to 0.2 micron absolute, and ozonation. A second water sample(USA II) was also obtained from the U.S. A third water sample (BP I) wasobtained from Budapest, Hungary. Capillary electrophroresis revealedsimilar ionic components differed in concentration between the threewaters.

The following cyclodextrins were added to the above described watersamples at a concentration range of 0.1%-5% w/v:

-   -   α-cyclodextrin (αCD also denoted as ACD), Lot. No. CYL-2322.    -   β-cyclodextrin (βCD also denoted as BCD). Lot. No. CYL-2518/2.    -   γ-cyclodextrin (γCD also denoted as GCD), Lot. No. CYL-2323.    -   2-hydroxypropyl-β-cyclodextrin (HPβCD, HPBCD), DS*=3.5, Lot. No.        CYL-2232.    -   2-hydroxypropyl-γ-cyclodextrin (HPCD, HPGCD), DS*=4.8, Lot. No.        CYL-2258.    -   carboxymethyl-β-cyclodextrin (CMBCD), Lot. No. CYL-2576.    -   quaternary-ammonium-β-cyclodextrin (QABCD).

For some examples, various inclusion complexes were formed betweencyclodextrins and complex-forming bioactive agents, including the aminoacids L-arginine and L-carnitine and the vitamin niacinamide (also knownas nicotinamide). All reagents were of analytical purity. For someexamples, L-arginine and nicotinamide were added in free form andalternatively in a cyclodextrin-complexed (molecularly entrapped) formto assess independent and co-dependent activities of a cyclodexrin and abioactive agent. Concentrations of above additives in free form, and ascyclodextrin inclusion complex forms, were in the range of 0.1% to 5.0%w/v.

Observations:

FIG. 7 shows second-derivative NIR spectra for the wavelength region900-1200 nm. The results show water-bond interactions are significantlymodified by addition of QABCD, and further significantly modified byaddition of CMBCD and HPBCD.

FIG. 8 shows second-derivative NIR spectra shown for 1200-1500 nm. Theresults show water-bond interactions are significantly modified byaddition of QABCD and HPBCD, and further significantly modified byaddition of CMBCD.

FIG. 9 shows second-derivative NIR spectra shown for 1620-1710 nm. Theresults show water-bond interactions are significantly modified byaddition of CMBCD, QABCD, and HPBCD.

FIG. 10 shows second-derivative NIR spectra shown for 2170-2370 nm. Theresults show water-bond interactions are significantly modified byaddition of CMBCD and HPBCD, and further significantly modified byaddition of QABCD.

As shown in FIGS. 7-10, addition of cyclodextrins alters molecularbonding interactions of the aqueous medium. Referring particularly toFIG. 9, refined NIR spectra derivatives in the wavelength range of1620-1770 nm show the carbon hydrogen bond related alterations involveCH3-CH2- and CH— groups of cyclodextrin additives. The significantspectral changes occurring in each cyclodextrin-treated water sampleindicate the modified micro-structure of hydrogen bonds governed clustersystems in bulk water. This effect was largest in the water samplestreated with charged quaternary-ammonium-β-cyclodextrins (QABCD), asshown for example in FIGS. 9 and 10.

Example 3 Acceleration of Plant Embryo Germination

Wheat seeds (Triticum aestivum) were germinated using USA I, USA II, andBP I waters described for Example 2. Germination rate usingun-supplemented (control) water was compared to that with the same watervariously supplemented with a cyclodextrin component, and/or a bioactiveagent, as an active component of cellular hydration. For each condition,ten seeds were placed in continuous water contact in a Petri-type dishkept at 25 C in 12 hr light/dark cycles. Photometric images wererecorded on days 1 to 6 after seeding. The percentage of seedsgerminated was calculated and compared as a function of time and of theapplied additive concentrations.

Water samples for seed germination were used alone with no additive, orcontaining cyclodextrins, or containing clathrate inclusion complexes ofcyclodextrin with L-arginine or with nicotinamide (both obtained fromSigma Chemical Co.; St. Louis, Mo.), or with L-carnitine (from Lonza AG;Switzerland). Additives were included at 0.1 and 5. % (w/v). Additivesolutions were prepared fresh on the day of germination start.

Parent cyclodextrins α-cyclodextrin (ACD), β-cyclodextrin (BCD), andγ-cyclodextrin (GCD), were obtained from Wacker Chemie (Munich,Germany). The following derivatized cyclodextrins were obtained fromCyclolab Ltd. (Budapest, Hungary): hydroxypropylated-beta-cyclodextrin(DS˜3) (HPBCD), carboxymethylated-β-cyclodextrin (DS˜3.5) (CMBCD),2-hydroxy-3-N,N,N-trimethylamino)propyl-β-cyclodextrin chloride (DS˜3.6)(QABCD).

Observations

Germination kinetics in control and additive-modified water underidentical conditions were quantified as the percentage of the seedshaving a sprout. Each determination consisted of 100 seeds for eachparameter. Results are reported in Table 1, below, and in FIGS. 11-13.

A) Cyclodextrin/L-Arg Inclusion Complex Increases Seed Germination.

TABLE 1 Effect of α-cyclodextrin and L-arginine on wheat seedgermination rate (values = percentage of total seeds) controlα-Cyclodextrin, α-CD/L-Arg Days (water) 0.5% L-Arg, 0.5% inc. complex 00 0 0 0 1 8 0 0 15 2 44 48 30 73 3 60 70 45 94 4 90 85 60 97

Table 1 shows comparative effects on the germination of wheat seeds of0.5% w/v α-CD, 0.5% w/v L-arginine (L-Arg), and 0.5% w/v of anα-CD/L-arginine inclusion complex, each dissolved in USA I water. Theabove-tabulated results indicate that, compared to pure water lackingany additive (control), wheat seed germination rate is much higher inwater including 0.5% (w/v) inclusion complex between α-cyclodextrin andL-arginine (αCD/L-Arg inc. complex). In addition, the results in Table 1indicate that wheat seed germination rate is much higher in waterincluding inclusion complex between α-cyclodextrin and L-arginine(αCD/L-Arg inc. complex) compared to water including 0.5% (w/v)α-cyclodextrin (αCD) as an additive alone, and also compared to waterincluding 0.5% (w/v) L-arginine (L-Arg) as an additive alone. Thus, theresults indicate a complex of α-cyclodextrin and L-arginine has asynergistic effect on increasing seed germination rate, which is notshown by either individual component of the complex used as a solitaryadditive. Results of Table 1 are also shown in FIGS. 11 and 13.

B) Cyclodextrin/Nicotinamide Inclusion Complex Increases SeedGermination.

TABLE 2 Effect of α-cyclodextrin and nicotinamide on wheat seedgermination rate Control α-Cyclodextrin, nicotinamide, α-CD/nicot. Days(water) 0.5% 0.5% inc. complex 0 2 0 0 0 1 9 11 0 0 2 50 18 4 65 3 62 6810 88 4 90 92 60 100

Table 2 shows comparative effects on the germination of wheat seeds of0.5% w/v α-cyclodextrin, 0.5% w/v nicotinamide, and 0.5% w/v of anα-cyclodextrin/nicotinamide inclusion complex (αCD/nicot. inc. Complex),each dissolved in USA I water. The above-tabulated results indicatethat, compared to pure water lacking any additive (control), wheat seedgermination rate is much higher in water including inclusion complexbetween α-cyclodextrin and nicotinamide. In addition, the results inTable 2 indicate that wheat seed germination rate is much higher inwater including inclusion complex between α-cyclodextrin andnicotinamide (αCD/nicot. inc. complex) compared to water includingα-cyclodextrin (αCD) as an additive alone, and also compared to waterincluding nicotinamide as an additive alone. Thus, the results indicatethat when used as an inclusion complex, α-cyclodextrin and nicotinamidehave a synergistic biological activity that significantly increases seedgermination rate. Such biological activity was not demonstrated byeither individual component of the complex used as a solitary additive.Results of Table 2 are also shown in FIGS. 12 and 13.

C) Qualitatively similar results as those reported in Tables 1 and 2,and FIGS. 11-13, were obtained using USA II and BP I water forgermination. Thus, in particular, cyclodextrin inclusion complexescontaining L-arginine, or alternatively containing nicotinamide, whendissolved in USA II or alternatively in BP I water, each significantlyincreased wheat seed germination rate, as shown above using USA I water.

D) Lengths of sprouts (rate of sprout growth during germination) did notdiffer between conditions within a statistically significant confidenceinterval (P<0.05). This result indicates that cyclodextrins, andparticularly cyclodextrin inclusion complexes, may be used selectivelyas active components of cellular hydration to promote a rate of seedgermination without necessarily also affecting a sprout growth rate.

Example 4 Lifespan Extension of C. Elegans in Hydration Modified Water

C. elegans nematodes were grown in petri-type dishes containing normalnutrient liquid media prepared alternatively with USA I water (describedin Example 2) lacking any further additive component (control) or thesame water supplemented with a parent α-, β-, or γ-cyclodextrin, and/ora bioactive agent, as an active component of cellular hydration. Fifty±3worms were transferred to each dish. Each condition was repeated intriplicate. Experiments were repeated for USA II and BP I watersdescribed in Example 2.

Water Additives:

A. Addition of parent α-, β- and γ-cyclodextrins.B. Addition of L-arginine and nicotinamide.C. Addition of inclusion complexes of cyclodextrins with L-arginine andnicotinamide.

Observations:

The results recorded are displayed below in Tables 3-5 and furtherpresented in FIGS. 14-18.

TABLE 3 Effect of cyclodextrins on C. elegans longevity Animals alive, %of initial (N = 50) Life Span Control α-Cyclodextrin, β-Cyclodextrin,γ-Cyclodextrin, (days) (water) 0.1% 0.1% 0.1% 10 92 100 100 100 15 10 2018 13 18 0 2 0 2

Table 3 reports the percentage of animals surviving to midlife (10days), advanced age (15 days) and old age (18 days), in media variablycontaining a parent α-, β-, and γ-cyclodextrin as an active component ofcellular hydration. In this example, parent cyclodextrins were added ata concentration of 0.1% w/v to nutritive media dissolved in USA I water.

Consistent with all previous studies, normal C. elegans animals in thepresent example survived two weeks in normal media. Each of the parentcyclodextrins markedly increased C. elegans survival (percentage alive)at advanced lifespan ages (days 10-15). Further, α-cyclodextrin andγ-cyclodextrins significantly increased the number of animals survivingto old ages, i.e., after day 15. The results are also representedgraphically in FIG. 14, which compares the cumulative percentages ofanimals surviving to 15 and 18 days in media containing each additiveparent cyclodextrin. The results show parent cyclodextrins, particularlyα- and β-cyclodextrin, may be used as an active component of cellularhydration to improve biological function in a live animal.

Biological mechanisms supporting advanced aging may include improvementof broad spectrum cellular activity during aging, or alternatively byselectively activating slow-aging cellular activity pathways.Clathrate-induced increases in water structure, hydration of cellularcomponents, and diffusivity of bioactive cellular components, includinginter- and intra-cellular signals, may all contribute to the overalleffects of cyclodextrins on organism survival.

TABLE 4 Effect of chemically-modified cyclodextrins C. elegans longevityAnimals alive, % of initial (N = 50) Quaternary- Life Span Control HP-β-Carboxymethyl- ammonium- (days) (water) Cyclodextrin β-Cyclodextrinβ-Cyclodextrin 10 90 96 94 98 15 8 17 11 13 18 0 0 0 2

Table 4 reports the percentage of animals surviving to midlife (10days), advanced age (15 days) and old age (18 days), in media variablycontaining a derivatized α-, β-, and γ-cyclodextrin as an activecomponent of cellular hydration. In this example, derivatizedcyclodextrins were added at 0.1% w/v to nutritive media dissolved in USAI water.

HP-, carboxymethyl-, and quaternaryammonium-derivatives ofβ-cyclodextrin had only slight effect on the initial survival of C.elegans to 10 days, as listed in Table 4. In contrast, significantincreases in survival were observed at advanced ages (15 days), but notat old ages (18 days). The results are also shown graphically in FIG.15, which compares the cumulative percentages of animals surviving to 15and 18 days in media containing each additive derivatized cyclodextrin.The results indicate derivatized cyclodextrins may be used as an activecomponent of cellular hydration to improve biological function in a liveanimal.

TABLE 5 Effect of cyclodextrin complexes on C. elegans longevity Animalsalive, % of initial (N = 50) Life Span Control α-CD/L- (days) (water)α-CD/L-Arg carnitine α-CD/nicotinamide 10 94 97 98 100 14 9 22 10 24 180 2 0 3

Table 5 reports the percentage of animals surviving to midlife (10days), advanced age (14 days), and old age (18 days), in nutritive mediadissolved in USA I water variably supplemented with a cyclodextrininclusion complex at 0.1% w/v, as an active component of cellularhydration. In this example, inclusion complexes contained α-cyclodextrinand a bioactive agent, particularly L-arginine, L-carnitine, orniacinamide.

As in previous examples, C. elegans animals in unsupplemented mediasurvived two weeks. α-Cyclodextrin complexes with L-arginine andniacinamide more than doubled C. elegans survival at advanced ages (day14), and further permitted a small but significant number of animals tosurvive to an old age, to which no animal survived in nutritive mediaalone. In contrast, α-cyclodextrin complexes with L-carnitine had littleor no significant effect on C. elegans survival. Similarly, L-arginineand nicotinamide added alone to the culture media without α-cyclodextrinhad little effect on C. elegans survival. Results are also showngraphically in FIG. 16, which compares the cumulative percentages ofanimals surviving to 14 and 18 days in media containing eachcyclodextrin inclusion complex as an additive. The results indicateα-cyclodextrin inclusion complexes, particularly complexes withL-arginine and niacinamide, may be used as an active component ofcellular hydration to improve biological function in a live animal.

As further shown in FIG. 17, an inclusion complex of α-cyclodextrin andL-arginine (data series A; 1:1 complex, dissolved at 0.1% w/v in mediamade with USA I water), and an inclusion complex of α-cyclodextrin andniacinamide (data series B; 1:1 complex, dissolved at 0.1% w/v in mediamade with USA I water) can decrease the mortality rate of C. elegansworms. FIG. 17 shows the number of animals dying on each day for eachmedia condition, wherein the control data series is media made with USAI water and lacking a further additive or supplement. The results showcomplexed forms of α-cyclodextrin may be used as an active component ofcellular hydration to retard mortality of a live animal.

FIG. 18 alternatively represents the data of FIG. 17 as a survival curvefor animals growing in normal media using USA I water (Control), oralternatively in media supplemented with a 1:1 inclusion complex ofα-cyclodextrin and L-arginine (Sample 1); or in media supplemented witha 1:1 inclusion complex of cyclodextrin and niacinamide (Sample 2).Thus, the delay in mortality shown in FIG. 17 results in an older age ofsurvival, the average age of survival (50% survival) increasing fromnearly 13 days in normal media to nearly 14 days in media including acyclodextrin inclusion complex as an active component of cellularhydration, which represents an 8% increase in lifespan.

Although the present invention has been shown and described withreference to the foregoing operational principles and preferredembodiments, it will be apparent to those skilled in the art thatvarious changes in form and detail may be made without departing fromthe spirit and scope of the invention. The present invention is intendedto embrace all such alternatives, modifications and variances that fallwithin the scope of the appended claims.

1. A composition that interacts with a biological cell system thatincludes bioactive molecules with biomolecular surfaces, cellularcomponents and water molecules with a specific density, comprising: abiologically active component that is constructed to increase anactivity of a biological cell system by increasing the hydration of oneor more components of that cell system.
 2. The composition of claim 1,wherein the biologically active component includes a primarycarbohydrate clathrate subcomponent that increases the H-bondedstructure of water.
 3. The composition of claim 2, wherein thebiologically active component includes a secondary solute subcomponent.4. The composition of claim 3, wherein the secondary solute subcomponentis a bioactive agent.
 5. The composition of claim 1, wherein thebiologically active component includes an inclusion complex that is madeup of a clathrate component and a complex-forming compound.
 6. Thecomposition of claim 5, wherein the complex-forming compound is abioactive agent.
 7. The composition of claim 2 wherein the clathratesubcomponent includes a compound chosen from the group consisting ofamyloses and cyclodextrins.
 8. The composition of claim 2 wherein theclathrate subcomponent includes a cyclodextrin.
 9. The composition ofclaim 8 wherein the cyclodextrin is constructed to exhibit kosmotropeactivity that increases the bonded structure of the water.
 10. Thecomposition of claim 9, wherein the cyclodextrin is constructed to causean increase in H-bonding between the water molecules, which increasemodifies the hydration of biomolecular surfaces of the biological cellsystem and thereby alters the interaction between cellular components ofthe biological cell system.
 11. The composition of claim 9, wherein thecyclodextrin is constructed to strengthen H-bonding between watermolecules, to cause an open water structure with a lower specificdensity, and to increase a rate of diffusion of the bioactive molecules.12. The composition of claim 11, further including bioactive moleculesthat are chosen from the group consisting of enzymes, enzyme substrates,nutrients, metabolites, cytokines, neurotransmitters, hormones,extracellular signals, intracellular messengers, and pharmacologicalagents.
 13. The composition of claim 7, wherein the cyclodextrin ischosen from the group consisting of α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, methylated α-, β-, and γ-cyclodextrins,2-hydroxypropylated β-cyclodextrins, water soluble β-cyclodextrinpolymers, partially acetylated α-, β-, and γ-cyclodextrins, ethylatedα-, β-, and β-cyclodextrins, carboxy-alkylated β-cyclodextrins,quaternary-ammonium salts of α-, β-, and γ-cyclodextrins, an amylose(e.g., an acetylated amylose), and mixtures thereof.
 14. The compositionof claim 7, further including a complex-forming agent, and wherein theclathrate subcomponent is capable of forming an inclusion complex withthe complex-forming agent.
 15. The composition of claim 14, wherein thecomplex-forming agent is chosen from the group consisting of aminoacids, vitamins, flavorants, odorants, and colorants.
 16. Thecomposition of claim 15, wherein the clathrate subcomponent is chosenfrom the group consisting of α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, 2-hydroxypropyl-cyclodextrins,caboxymethylated-cyclodextrins, quaternary-ammonium-cyclodextrins,amyloses, amylose derivatives, and mixtures thereof.
 17. A compositionthat interacts with a biological cell system that includes bioactivemolecules with biomolecular surfaces, cellular components and watermolecules with a specific density, comprising: a primary carbohydrateclathrate component that is capable of increasing the H-bonded structureof water; a secondary solute subcomponent; and wherein the clathratecomponent and the solute component are capable of increasing an activityof a biological cell system by increasing the hydration of one or morecomponents of that cell system.
 18. The composition of claim 17, whereinthe clathrate subcomponent includes a compound chosen from the groupconsisting of amyloses and cyclodextrins.
 19. The composition of claim17, wherein the secondary solute subcomponent is a bioactive agent. 20.The composition of claim 17, further including a complex-formingcompound, and wherein the clathrate compound and the complex-formingcompound form an inclusion complex.
 21. The composition of claim 20,further including bioactive molecules that are chosen from the groupconsisting of enzymes, enzyme substrates, nutrients, metabolites,cytokines, neurotransmitters, hormones, extracellular signals,intracellular messengers, and pharmacological agents.
 22. Thecomposition of claim 18, wherein the clathrate component is acyclodextrin that is chosen from the group consisting of α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, methylated β-cyclodextrins,2-hydroxypropylated β-cyclodextrins, water soluble β-cyclodextrinpolymers, partially acetylated α-, β-, and γ-cyclodextrins, ethylatedα-, β-, and β-cyclodextrins, carboxy-alkylated β-cyclodextrins,quaternary-ammonium salts of α-, β-, and γ-cyclodextrins, an amylose(e.g., an acetylated amylose), and mixtures thereof.
 23. The compositionof claim 22, wherein the complex-forming agent is chosen from the groupconsisting of amino acids, vitamins, flavorants, odorants, andcolorants.
 24. The composition of claim 18, wherein the clathratesubcomponent is chosen from the group consisting of α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-cyclodextrins,caboxymethylated-cyclodextrins, quaternary-ammonium-cyclodextrins,amyloses, amylose derivatives, and mixtures thereof.
 25. A beverage thatinteracts with a biological cell system that includes bioactivemolecules with biomolecular surfaces, cellular components and watermolecules with a specific density, comprising: an aqueous componentchosen from the group consisting of still and carbonated liquids; aprimary carbohydrate clathrate component that is capable of increasingthe H-bonded structure of water; a secondary solute subcomponent; andwherein the clathrate component and the solute component are capable ofincreasing an activity of a biological cell system by increasing thehydration of one or more components of that cell system.
 26. Thebeverage of claim 25, wherein the clathrate subcomponent includes acompound chosen from the group consisting of amyloses and cyclodextrins.27. The beverage of claim 26, wherein the secondary solute subcomponentis a bioactive agent.
 28. The beverage of claim 27, further including acomplex-forming compound, and wherein the clathrate compound and thecomplex-forming compound form an inclusion complex.
 29. The beverage ofclaim 28, further including a flavorant that is chosen from the groupconsisting of apple, apricot, banana, grape, blackcurrant, raspberry,peach, pear, pineapple, plum, orange, and vanilla flavorants. Examplesof flavorant related compounds include butyl acetate, butyl isovalerate,allyl butyrate, amyl valerate, ethyl acetate, ethyl valerate, amylacetate, maltol, isoamyl acetate, ethyl maltol, isomaltol, diacetyl,ethyl propionate, methyl anthranilate, methyl butyrate, pentyl butyrate,pentyl pentanoate, erythritol, maltitol, mannitol, sorbitol, lactitol,xylitol, inositol, isomalt, propylene glycol, glycerol (glycerine),threitol, galactitol, palatinose, reduced isomalto-oligosaccharides,reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reducedmaltose syrup, and reduced glucose syrup.
 30. The beverage of claim 28,further including a colorant that is chosen from the group consisting ofbetalains, betacyanins, betaxanthins, vulgaxanthin, miraxanthin,portulaxanthin, indicaxanthin, anthocyanidins, aurantinidin, cyanidin,delphinidin, europinidin, luteolinidin, pelargonidin, malvidin,peonidin, petunidin, rosinidin, corresponding anthocyanins or glucosidesof anthocyanidins, turmeric type colorants, phenolic curcuminoids,curcumin, demethoxycurcumin and bisdemethoxycurcumin.
 31. The beverageof claim 28, further including a vitamin that is chosen from the groupconsisting of nicotinamide (vitamin B₃), niacinamide, niacin, pyridoxalhydrochloride (vitamin B₆), ascorbic acid, edible ascorbyl esters,riboflavin, pyridoxine, thiamine, vitamin B₉, folic acid, folate,pteroyl-L-glutamic acid, pteroyl-L-glutamate, salts thereof, andmixtures thereof.
 32. The beverage of claim 28, further including anamino acid or amine.
 33. The beverage of claim 32, wherein the aminoacid is chosen from the group consisting of aspartic acid, arginine,glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine,alanine, valine, tyrosine, leucine, isoleucine, asparagine, serine,lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid(alpha-, beta-, and gamma-isomers), glutamine, hydroxyproline, taurine,norvaline, sarcosine, salts thereof, mixtures thereof, and N-alkyl C₁-C₃and N-acylated C₁-C₃ derivatives of these amino acids, and mixtures ofany of the amino acids or derivatives thereof.
 34. A method of improvingcellular hydration in an animal, such as a human, with a body in whichphysiological conditions occur, and in which there is a biological cellsystem that includes bioactive molecules with biomolecular surfaces,cellular components and water molecules with a specific density,comprising: preparing a beverage by dissolving an inclusion complexformed by a carbohydrate clathrate component and a complex-forming agentcapable of dissociating from carbohydrate clathrate component under thephysiological conditions; and causing the animal to orally ingest thebeverage, whereupon the carbohydrate clathrate component dissociatesfrom the complex-forming agent and modifies the strength, extent, andkinetics of the hydrogen bonded water structure at cellular biomolecularsurfaces.
 35. The method of claim 34, further including the step ofchoosing the clathrate component from the group consisting of amylosesand cyclodextrins.
 36. The method of claim 35, wherein the choosing stepincludes choosing a cyclodextrin from the group consisting ofα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methylatedβ-cyclodextrins, 2-hydroxypropylated β-cyclodextrins, water solubleβ-cyclodextrin polymers, partially acetylated α-, β-, andγ-cyclodextrins, ethylated α-, β-, and β-cyclodextrins,carboxy-alkylated β-cyclodextrins, quaternary-ammonium salts of α-, β-,and γ-cyclodextrins, an amylose (e.g., an acetylated amylose), andmixtures thereof.
 37. The method of claim 34, further including the stepof selecting a complex-forming agent from the group consisting of aminoacids, vitamins, flavorants, odorants, and colorants.